Mouse neuroblastoma cells (N2a) were grown at 37C in a humidified incubator chamber under 5% CO2 in OptiMEM (Gibco), supplemented with 5% fetal bovine serum (FBS) (Atlanta Biologicals), 1% penicillin, and 1% streptomycin (Gibco). Cells were plated into 8-well chamber slides (Sarstedt) at a density of 30,000 cells/well and transiently transfected using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s instructions and imaged 24 h later.
Primary cortical neurons were prepared as previously described [8 (link)]. Briefly, neurons were obtained from embryonic day 14 (E14) CD1 (Charles Rives Laboratories) mouse embryos. Neurons were dissociated using Papain dissociation system (Worthington Biochemical Corporation, Lakewood, NJ, USA). Cells were plated in 8-well chamber slides previously coated with poly-D-lysine at a density of 30,000 cells/well and were maintained for 10–14 days in vitro (DIV) in Neurobasal medium supplemented with 2% B27 (Gibco), 1% penicillin/streptomycin (Gibco) and 1% glutamax (Gibco) in a humidified 37 C incubator with 5% CO2 without further media exchange. Neurons were either transfected using Lipofectamine 2000 (Life Technologies) or infected with AAV.hSyn.mt-roGFP after 12–14 DIV, and imaged 1 or 3 days later respectively. Experiments were performed after a culturing period of 12–14 DIV.
Primary cortical neurons were prepared as previously described [8 (link)]. Briefly, neurons were obtained from embryonic day 14 (E14) CD1 (Charles Rives Laboratories) mouse embryos. Neurons were dissociated using Papain dissociation system (Worthington Biochemical Corporation, Lakewood, NJ, USA). Cells were plated in 8-well chamber slides previously coated with poly-D-lysine at a density of 30,000 cells/well and were maintained for 10–14 days in vitro (DIV) in Neurobasal medium supplemented with 2% B27 (Gibco), 1% penicillin/streptomycin (Gibco) and 1% glutamax (Gibco) in a humidified 37 C incubator with 5% CO2 without further media exchange. Neurons were either transfected using Lipofectamine 2000 (Life Technologies) or infected with AAV.hSyn.mt-roGFP after 12–14 DIV, and imaged 1 or 3 days later respectively. Experiments were performed after a culturing period of 12–14 DIV.
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